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首页> 外文期刊>RSC Advances >Protein binding-protected DNA three-way junction-mediated rolling circle amplification for sensitive and specific detection of transcription factors
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Protein binding-protected DNA three-way junction-mediated rolling circle amplification for sensitive and specific detection of transcription factors

机译：受蛋白质结合保护的DNA三元连接介导的滚动圆扩增，用于敏感和特异性检测转录因子

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Transcription factors (TFs) are DNA-binding proteins that regulate gene transcription and their expression levels are closely associated with disease development. Sensitive and specific detection of TFs is significant to clinical diagnostics and drug development. Herein, a label-free fluorescent strategy for the sensitive and specific detection of TFs was developed based on protein binding-protected DNA three-way junction (TWJ)-mediated rolling circle amplification (RCA). A trifunctional TWJ was designed including a target binding site, AlwI recognition site and sutured primer of RCA. Firstly, TFs bound with a target binding site, protecting the four and five bases downstream from the AlwI recognition site against cleavage by AlwI. Next, the sutured primer in the protected TWJ hybridized with the padlock probe, initiating RCA. Finally, the sutured primer was extended with multiple G-quadruplex sequences, binding with N -methyl-mesoporphyrin IX (NMM) to yield an enhanced fluorescence response. Residual TWJs were digested by AlwI, effectively blocking the RCA reaction, thus suppressing nonspecific amplification. Taking NF-κB p50 as a model target of TFs, high sensitivity was achieved with a low detection limit of 6.8 pM and a broad linear range from 8 pM to 15 nM. We successfully measured NF-κB p50 in HeLa cell nuclear extracts with a low detection limit of 0.34 ng μL ~(?1) . The strategy was also effectively used to assay the inhibition effect of a model inhibitor of NF-κB, oridonin. The results indicated the proposed strategy holds great promise for studying TFs in disease diagnostics and drug developments.
机译：转录因子（TFS）是调节基因转录的DNA结合蛋白质，其表达水平与疾病发育密切相关。对TFS的敏感和特异性检测对于临床诊断和药物开发具有重要意义。在此，基于蛋白质结合保护的DNA三路接线（TWJ）介导的滚动圆扩增（RCA）开发了用于敏感和特异性TFS的无特异性检测的无标记荧光策略。设计了一个三官能的TWJ，包括目标结合位点，Alwi识别位点和RCA的缝合底漆。首先，与靶结合位点结合的TFS，保护来自Alwi识别部位下游的四个和五个基础反对Alwi的切割。接下来，在受保护的TWJ中的缝线引物与挂锁探针杂交，启动RCA。最后，缝合底漆用多个G-四边形序列延伸，与N-甲基 - 中卟啉IX（NMM）结合，得到增强的荧光反应。通过Alwi消化残留的TWJS，有效地阻断RCA反应，从而抑制非特异性扩增。服用NF-κBP50作为TFS的模型靶标，通过低检测限为6.8μm的低灵敏度，并且从8pm至15nm的宽线性范围。我们在Hela细胞核提取物中成功测量了NF-κBP50，检测限为0.34ngμl〜（α1）。该策略还有效地用于测定NF-κB，奥胺素模型抑制剂的抑制作用。结果表明，拟议的策略对研究疾病诊断和药物发展的TFS具有很大的承诺。

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